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Abstract #52053 Published in IGR 14-4
Bicarbonate-dependent secretion and proteolytic processing of recombinant myocilin
Aroca-Aguilar JD; Martínez-Redondo F; Martín-Gil A; Pintor J; Coca-Prados M; Escribano JPLoS ONE 2013; 8: e54385
Myocilin is an extracellular glycoprotein of poorly understood function. Mutations of this protein are involved in glaucoma, an optic neuropathy characterized by a progressive and irreversible visual loss and frequently associated with elevated intraocular pressure. We previously showed that recombinant myocilin undergoes an intracellular proteolytic processing by calpain II which cleaves the central region of the protein, releasing one N- and one C-terminal fragment. Myocilin cleavage is reduced by glaucoma mutations and it has been proposed to participate in intraocular pressure modulation. To identify possible factors regulating the proteolytic processing of recombinant myocilin, we used a cellular model in which we analyzed how different culture medium parameters (i.e., culture time, cell density, pH, bicarbonate concentration, etc.) affect the presence of the extracellular C-terminal fragment. Extracellular bicarbonate depletion associated with culture medium acidification produced a reversible intracellular accumulation of full-length recombinant myocilin and incremented its intracellular proteolytic processing, raising the extracellular C-terminal fragment percentage. It was also determined that myocilin intracellular accumulation depends on its N-terminal region. These data suggest that aqueous humor bicarbonate variations could also modulate the secretion and cleavage of myocilin present in ocular tissues.
Laboratorio de Genética Molecular Humana, Facultad de Medicina/Instituto de Investigación en Discapacidades Neurológicas-IDINE, Universidad de Castilla-La Mancha, Albacete, Spain.
Full articleClassification:
3.4.2 Gene studies (Part of: 3 Laboratory methods > 3.4 Molecular genetics)
3.6 Cellular biology (Part of: 3 Laboratory methods)